Drosha非依赖性miR-6778-5p调控胃癌干细胞干性的机制研究

Gastric cancer (GC)is a malignant cancer with poor outcome and no effective therapeatic target. Gastric cancer stem cell(GCSC) plays a critical role for tumor recurrence, metastasis and drug resistance.So far the mechanism of GCSC stemness maintence is still unknown. Our previous work shows that silenced Drosha does not completely reverse the malignant behavior of gastric cancer, and that increased Drosha-independent miRNAs are identified in Drosha silenced gastric cancer by microarray. Bioinformatics analysis reveales that MiR-6778-5p, which derived from the intron of SHMT1 gene, may closely impact GCSC formation and stemness, and regulates SHMT1 expression at a feedback pattern.How to regulate formation and characteristics of GCSC by miR-6778-5p and by SHMT1 has not been reported. In this project, molecular biology analyses will be conducted to explore the relationship between SHMT1,miR-6778-5p expression and gastric cancer stem cell (GCSC) and malignancy of GC. We will further uncover the mechanism of miR-6778-5p in regulating SHMT1 expression or its activation, and analyze metabolites changes of one carbon metabolism mediated by SHMT1 and the effect on GCSC formation and stemness. The results may be helpful to understand a novel mechanism of GCSC stemness maintenance regulated by Drosha-independent miRNA, which will provide a new target for gastric cancer therapy through blockage of one carbon metabolism.

胃癌恶性程度高预后差，尚缺乏有效治疗靶标。胃癌干细胞（GCSC）是胃癌复发转移及耐药根源，其干性调控机制的研究仍不充分。我们前期发现Drosha沉默未完全逆转胃癌恶性行为，芯片及预实验鉴定到一系列高表达的Drosha非依赖性miRNAs，生物信息分析发现SHMT1基因内含子剪切来源的miR-6778-5p与GCSC密切、且反馈调节SHMT1。miR-6778-5p及一碳代谢关键酶SHMT1如何调控GCSC干性未见报道。本项目拟进一步采用分子生物学方法，验证SHMT1、miR-6778-5p与GCSC干性及胃癌恶性程度关联性；研究SHMT1内含子剪切来源的miR-6778-5p，对SHMT1表达/活性反馈调控，探究SHMT1介导的一碳代谢产物变化及其对GCSC干性维持的作用。研究结果将揭示GCSC干性维持新机制，为靶向能量代谢干预GCSC的胃癌治疗提供新靶标。

Drosha依赖的经典miRNA在癌症的生物学功能和发展中发挥着至关重要的作用。然而，不依赖Drosha的非经典miRNA的功能仍然知之甚少。前期我们在Drosha敲低的胃癌(GC)细胞中发现了一组异常上调的非经典miRNA，被称为Drosha非依赖性miRNA。令人惊讶的是，Drosha沉默的GC细胞仍然保留了强烈的恶性特性（例如，增殖迁移能力和肿瘤干细胞(CSC)特征），表明异常上调的非经典miRNA可能在维持低Drosha水平胃癌细胞的恶性特性中有重要作用。. 我们报告了一种非经典miR6778-5p，在Drosha敲低GC细胞中充当维持CSC干性的关键调节因子。MiR6778-5p属于5'-尾mirtron类miRNA，是SHMT1（编码细胞质丝氨酸羟甲基转移酶）的内含子5的转录剪接产物，通过靶向YWHAE正向反馈调节其宿主基因SHMT1的表达，SHMT1催化叶酸依赖的丝氨酸/甘氨酸相互转化一碳代谢。有趣的是，正常细胞和大多数癌细胞中主要是SHMT2介导的线粒体一碳代谢，而在Drosha低表达胃癌细胞中SHMT2表达下调，miR6778-5p通过调控SHMT1介导的细胞质一碳代谢代偿性激活，从而在维持胃癌干细胞干性。高表达SHMT1的胃癌干细胞具有5-Fu抗性，miR6778-5p或SHMT1的缺失显著减少了GCSC的球体形成并增加了胃癌细胞对5-Fu的敏感性。.同时，我们揭示了另一非经典miR-4646-5p在胃癌转移中起关键作用。MiR-4646-5p是另一种不依赖Drosha的mirtron类miRNA，是宿主基因Abhd16a内含子3在SRSF2辅助下的特异性转录剪接产物。miR-4646-5p可以通过靶向PHD3增强HIF1A稳定性，从而正反馈调节ABHD16A和miR-4646-5p的表达。ABHD16A作为一种新兴的磷脂酰丝氨酸特异性脂肪酶参与脂质代谢，导致溶血磷脂酰丝氨酸（lyso-PSs）的积累，通过GPR34/Gi亚基刺激RhoA和下游LIMK/cofilin级联活性，促进胃癌的转移。. 本文研究非经典miRNAs在肿瘤进展中的新功能，可作为胃癌诊断新的生物标志物。miR-6778-5p及其宿主基因SHMT1介导的一碳代谢、MiR-4646-5p及Abhd16a介导的异常脂质代谢可能是胃癌临床治疗的新靶点。